Routine high-sensitivity multiplexed detection of disease biomarkers in blood will have an impact on early diagnosis and therapy selection. A novel and highly effective approach for detection of broad categories of disease is multiplexed detection of antibodies using protein microarrays. As a greater number of antibody biomarkers are being identified and clinically validated for viral infection, cancer, cardiovascular disease, and autoimmune disease, there is a strong need to detect them with greater sensitivity and greater signal-to-noise ratio, while at the same time being able to perform automated biomarker analysis with low cost per test. In the proposed project, the investigators integrate photonic crystal enhanced fluorescence (PCEF) technology with a novel size-exclusion blood filtration technology to develop a multiplexed microspot fluorescent sandwich assay platform for the clinical laboratory environment. Our approach integrates several innovations into an inexpensive plastic-based sensor cartridge and desktop detection instrument: 1. A silicon-based nanostructured resonant optical photonic crystal chip with an integrated Fabry-Perot optical cavity will be used to deliver ~50x greater signal enhancement than current-generation PCEF devices, which already routinely provide <1 pg/ml limits of detection in complex media, using only 10 ?l sample volumes. 2. A laser-machined blood filter will separate plasma from a droplet of heparinized whole blood in 60 seconds, enabling the entire assay protocol to be performed automatically in <60 minutes without user intervention. 3. An innovative laser scanning approach is used to couple light from a small semiconductor laser directed to the PC surface in the optimal ?on-resonance? condition, resulting in a rugged, compact instrument with a cost of <$10K. 4. A statistical bioinformatics tool indicating the presence or absence of the biomarkers in the test sample. As an example application of the system, we will focus our effort on detection of a panel of 3 serum antibodies for human papillomavirus (HPV) as a means for identifying patients with greater susceptibility to oropharyngeal carcinoma (OPC), although the same technology can be extended to detection of broad classes of antibody biomarkers. The new instrument will be first tested upon biomarkers spiked into whole blood, with results compared against single-antibody ELISA in microplates and the Luminex bead-based system. Further validation and comparison will be performed upon clinical blood samples from patients with known HPV exposure. Our long-term goal is to demonstrate a prototype system that can be applied broadly for multiplexed serum antibody biomarker analysis.